Simple In-Cell Processing Enables Deep Proteome Analysis of Low-Input Caenorhabditis elegans

Caenorhabditis elegans is a widely
used genetic model organism; however, the worm cuticle complicates
extraction of intracellular proteins, a prerequisite for typical bottom-up
proteomics. Conventional physical disruption procedures are not only
time-consuming but can also cause significant sample loss, making
it difficult to perform proteomics with low-input samples. Here, for
the first time, we present an on-filter in-cell (OFIC) processing
approach that can digest C. elegans proteins directly in the cells of the organism after methanol fixation.
With OFIC processing and single-shot LC-MS analysis, we identified
over 9400 proteins from a sample of only 200 worms, the largest C. elegans proteome reported to date that did not
require fractionation or enrichment. We systematically evaluated the
performance of the OFIC approach by comparing it to conventional lysis-based
methods. Our data suggest superior performance of OFIC processing
for C. elegans proteome identification
and quantitation. We further evaluated the OFIC approach with even
lower-input samples, including single worms. Then, we used this method
to determine how the proteome is impacted by loss of superoxide dismutase sod-1, the ortholog of human SOD1, a gene
associated with amyotrophic lateral sclerosis. Analysis of 8800 proteins
from only 50 worms as the initial input showed that loss of sod-1 affects the abundance of proteins required for stress
response, ribosome biogenesis, and metabolism. In conclusion, our
streamlined OFIC approach, which can be broadly applied to other systems,
minimizes sample loss while offering the simplest workflow reported
to date for C. elegans proteomics.